Takeya A, Hosomi O, Ishiura M
Department of Legal Medicine, Gunma University School of Medicine.
J Biochem. 1990 Mar;107(3):360-8. doi: 10.1093/oxfordjournals.jbchem.a123051.
Human alpha-3-N-acetylgalactosaminyltransferase has been purified 27,000,000-fold from A1 plasma by (NH4)2SO4 fractionation and affinity chromatography on Sepharose 4B, anti-human group O plasma antibodies-Sepharose 4B, and Blue Dextran-Sephadex G-25. A modified procedure in the Sepharose 4B step was developed by batch adsorption and desorption experiments. Cibacron Blue F3G-A, the chromophore of Blue Dextran, was found to bind to the enzyme. UDP is an effective inhibitor of this binding. The pure transferase has an apparent molecular weight of 35,000 as judged by SDS-PAGE in the presence of a reducing agent. The specific activity is 16 pmol/min.ng enzyme, which is comparable to that (30 pmol/min.ng enzyme) of alpha-3-N-acetylgalactosaminyltransferase from porcine submaxillary glands [Schwyzer and Hill (1977) J. Biol. Chem. 252, 2338-2355]. The apparent Km values for UDP-GalNAc, 2'-fucosyllactose, and lacto-N-fucopentaose I are 13, 270, and 350 microM, respectively. The reaction velocity was found to fall off again at high concentrations of oligosaccharide acceptor substrates. The apparent Ki values for UDP and UDP-galactose are 8.6 and 6.2 microM, respectively. The pure enzyme also catalyzes the transfer of galactose in alpha-linkage to 2'-fucosyllactose though the transfer rate of galactose is much lower than that of N-acetylgalactosamine.
人α-3-N-乙酰半乳糖胺基转移酶已通过硫酸铵分级分离以及在琼脂糖4B、抗人O型血浆抗体-琼脂糖4B和蓝色葡聚糖-葡聚糖凝胶G-25上的亲和层析从A1血浆中纯化了27000000倍。通过分批吸附和解吸实验开发了琼脂糖4B步骤中的改良方法。发现蓝色葡聚糖的发色团Cibacron Blue F3G-A与该酶结合。UDP是这种结合的有效抑制剂。在还原剂存在下通过SDS-PAGE判断，纯转移酶的表观分子量为35000。比活性为16 pmol/分钟·纳克酶，这与猪颌下腺的α-3-N-乙酰半乳糖胺基转移酶（30 pmol/分钟·纳克酶）相当[施维泽和希尔（1977年）《生物化学杂志》252，2338 - 2355]。UDP-GalNAc、2'-岩藻糖基乳糖和乳糖-N-岩藻戊糖I的表观Km值分别为13、270和350微摩尔。发现反应速度在高浓度的寡糖受体底物时再次下降。UDP和UDP-半乳糖的表观Ki值分别为8.6和6.2微摩尔。纯酶也催化α-连接的半乳糖转移到2'-岩藻糖基乳糖，尽管半乳糖的转移速率远低于N-乙酰半乳糖胺的转移速率。
